The major goal of Project 3 (William B. Uphold, PI) will be to investigate the transcriptional regulation of Msx2 expression in the developing vertebrate limb with particular emphasis on identification of cis-regulatory elements and trans-acting factors that control the independently-regulated spatially-specific domains of Msx2 expression in the AER and limb mesoderm. Two separate enhancer elements have been identified in the Msx2 gene that direct expression of reporter genes to the AER of transgenic mice. Reporter constructs in transgenic mice will be used to define further these two AER enhancer elements and to examine the functional interactions between the two sites. Similar approaches will be used to study the possibility that sequences at DNase hypersensitive sites identified in the Msx2 gene during the current period of support play a role in regulating the spatially-specific domains of Msx2 expression. The 1-hybrid screening process or the Southwestern screening technique will be used to identify candidate trans-acting molecules that interact with the enhancer regions identified in the Msx2 gene. The regulatory sequences involved in the response of the Msx2 gene to other regulatory genes such as Dlx5 and signaling such as Sonic hedgehog, BMP-4, IGF-1, and FGFs that are involved in regulating AER activity and/or limb patterning will also be investigated by determining the effect of these factors on the expression of a variety of Msx2 reporter gene constructs in cultured limb cells. In addition, the role of Msx2 in regulating the expression of the BMP-1, a putative downstream target of msx2 in the limb, will be studied in co-transfection experiments using Msx2 expression vectors and BMP4 promotor reporter gene constructs. These studies should provide insight into how the Msx gene is regulated and how the regulatory network is involved in pattern formation in the developing limb.